Cells in
culture or in vitro are a useful model for studying the activity of
cells in the whole organism or in vivo. Ten years ago or so cell
culture techniques were considered somewhat esoteric. Today because of our
better understanding of cell nutrition, metabolism and general growth
environment it has become a fairly routine procedure. Today's lab will
consist of subculturing and quantifying a vertebrate cell line derived from
one-day old zebrafish embryos and is called the ZF4 line.

The ZF4
line is a fairly prototypical cell line and behaves very much like many of
the mammalian lines that are available. The American Type Culture Collection
in Rockville, Maryland is one of the major repositories for the large number
of cell lines that are available.

To obtain a
given cell line you just send them the name of the cells you wish to culture
along with $425.00 and it arrives a few days later frozen on dry ice. These
cultures are then rapidly thawed and placed in their appropriate growth
medium under sterile conditions, and if all works out well you have a cell
line to work with. I received the ZF4 line in this manner last month. Some
characteristics of the cell line are given below.

9. Subcultivation Ratio:A subcultivation ratio of 1:2 to 1:4 is
recommended with media
renewal 2 to 3
times per week

I am
assuming you know how to use the compound microscope. If you have any
questions or need a review of its operation, feel free to ask. A specialized
type of microscope that is used in cell culture work is called an inverted
phase contrast microscope. These are expensive but easy to operate. The
light source is located above the stage and the objective lenses are located
below the stage.

The light
source and phase contrast optics take advantage of the refractive indexes of
live cells to give contrast to unstained specimens. We will be using this
microscope to monitor our cultures. There are a number of different
techniques available to perform routine cell culture work. Below are some
procedures I have found to work well.

II.
PROCEDURES

STERILE
TECHNIQUES

Aseptic or
sterile technique is the execution of tissue culture procedures without
introducing contaminating microorganisms from the environment. In doing
tissue culture work, 70% of the problems are due to a lack of good sterile
technique. Microor­ganisms causing the contamination problems exist
everywhere, on the surface of all objects and in the air.

A conscious
effort must be made to keep them out of a sterile environment. Because many
and sometimes awkward manipulations are required for various techniques,
tissue culture media used are often supple­mented with antibiotics.
Antibiotics do not eliminate problems of gross contamination which result
from poor sterile technique or antibiotic-resistant mutants. Autoclaving
renders pipettes, glass­ware, and solutions sterile.

Nutrient
medium cannot be autoclaved. The compounds in nutrient medium are destroyed
by the heat of autoclaving. Medium must therefore be sterilized by passing
it through a sterile filter small enough in pore size to hold back bacteria
and mycoplasmas (Millipore Sterivex - GS 0.22u disposable filter units).
Here are some rules of thumb to follow to keep your medium, cultures, and
glassware from becoming contaminated:

1. Wipe your work area
and hands with 70% ethanol before starting.

2. Never uncover a
sterile flask, bottle, petri dish, etc., until the instant you are ready to
use it. Return the cover as soon as you are finished. Never leave it open
to the environment.

3. Sterile pipettes
should never be taken from the wrapper until they are to be used. Keep your
pipettes at your work area. Sterile pipettes do not have to be flamed.
Pipetting your cells with a hot pipette will kill them.

4. When removing the cap
from a bottle, flask, etc., do not place the cap with the open end upright
on the lab bench. Do not hold the opening straight up into the air. If
possible, tilt the container so that any falling microorganisms fall onto
the lip.

5. Be careful not to
talk, sing, or whistle when you are per­forming these sterile procedures.

6. Do not draw from a
different bottles with the same pipette. Because such a pipette has been
exposed; the chance for contamination is too great; use sterile pipette for
each bottle -- especially when pipetting medium.

7. Techniques should be
performed as rapidly as possible to minimize contamina­tion.

You may
find yourself involved in a procedure which these sterile technique
"rules-of-thumb" do not cover. Therefore, you must constantly be aware that
microorganisms are everywhere and take proper steps to keep them out of your
cultures. When first developing your aseptic technique you must always be
thinking of sterility. Eventually it will become second nature to you.

Mastering
good aseptic technique will save you considerable frustration in the labs to
follow. Furthermore, the same princi­ples for good aseptic technique also
minimize biohazard risk to the investigator when infec­tious organisms or
dangerous chemicals are used.

"EYEBALLING" THE CULTURES

Before doing
anything with a culture, its general "health" and appearance should be
evaluated. This can be done quickly and quantitatively by making the following
observations:

1. Check the pH of the
culture medium by looking at the color of the indicator, phenol red. As a
culture becomes more acid the indicator shifts from red to yellow-red to
yellow. As the culture becomes more alkaline the color shifts from red to
fuchsia (red with a purple tinge). As a generaliza­tion, cells can tolerate
slight acidity better than they can tolerate shifts in pH above pH 7.6.

2. Cell attachment. Are
most of the cells well attached and spread out? Are the floating cells dividing
cells or dying cells which may have an irregular appear­ance?

3. Percent confluency. The
growth of a culture can be estimat­ed by following it toward the development of
a full cell sheet (confluent culture). By comparing the amount of space covered
by cells with the unoccupied spaces you can estimate percent confluency.

4. Cell shape is an
important guide. Round cells in an un­crowded culture is not a good sign unless
these happen to be divid­ing cells. Look for doublets or dividing cells. Get
to know the effect of crowding on cell shape.

5. Look for giant cells.
The number of giant cells will in­crease as a culture ages or declines in
"well-being." The frequency of giant cells should be relatively low and
con­stant under uniform culture conditions.

6. One of the most valuable
guides in assessing the success of a "culture split" is the rate at which the
cells in the newly established cultures attach and spread out. Attach­ment
within an hour or two suggests that the cells have not been traumatized
and that the in vitro environment is not grossly abnormal. Longer
attachment times are suggestive of problems. Nevertheless, good cultures may
result even if attachment does not occur for four hours.

7. Keep in mind that some
cells will show oriented growth patterns under some circumstances while many
transformed cells, because of a lack of contact inhibition may "pile up"
especially when the culture becomes crowded. Get to recog­nize the range of
cells shapes and growth patterns exhibit­ed by each cell line.

2. Optional: Rinse by
adding 7 ml of PBS or growth media without sera to the T-25, then decant into
waste collection jar.

3. Rinse by adding 3 ml of
cold trypsin to the T-25, then decant into waste collection jar. This will
remove the proteins present from the old media that tend to decrease the ability
of the trypsin to act on the cells.

4. Now add 3 ml of cold
trypsin and examine under low magnification for 3-7 minutes with the phase
contrast inverted scope. When it appears that most of the cells have rounded up,
but have not yet completely detached, whack the T-25 gently. Then immediately
return to the hood and pipette the cells up and down several times to help break
up the clumps of cells.

5.
Remove 0.1 ml (100 ul) of cells in trypsin and place in microfuge tube for cell
counting.

6. Add 9.0 ml fresh media to
three new flasks (if you are doing a 1:3 split). Using the same pipette, and as
continuation of the same process, draw up the cell suspension and quickly
dispense a 0.9 ml aliquot into each T-25 flask containing 9.1 ml of media in the
new flask giving a total volume of 10 ml.

7. Incubate at 28C and
monitor periodically, beginning 30-45 minutes after inoculation. Rapid
attachment (within 1 hour or so) is indicative, but not proof, that all went
well.

8. Start making
observations, such as those described in the handout "Eyeballing the Culture."

Directions
for Use of Coulter Counter Particle Counter

1.
Turn power switch to “I” position to turn instrument on

2.
Lower specimen tray and replace blue “clenz” fluid with salt solution then raise
up tray and place the aperture in salt solution

3.
Press “functions” till F1 screen comes up then use arrow keys (>) to select
“Prime Aperture”, then hit start, this takes you to F2 screen, then
press start again

4.
When finished, remove salt solution and replace with your sample in salt
solution, be sure that the aperture is in sample

9. At
the end of the day do several counts (4-8) with blue Coulter “Clenz” solution,
this is very important as it fills the aperture, pump and tubes in
system with blue cleansingsolution and prevents lockups.

10.
Every week replace blue Clenz fluid in cup and select “functions” then select
“fill system”, this replaces fluid in system and prevents lockups

HEMOCYTOMETER COUNTING AND CELL
VIABILITY (OPTIONAL)

The importance
of accurate enumeration of cells at time of inoculation, at the termina­tion of
an experiment, and throughout the course of many experiments is self-evident.
Cell enumeration with a hemocytometer is the most widely used method, and it
continues to have a place in all laboratories, including those equipped with
electronic cell counters (like us). This laboratory exercise will serve to
intro­duce the use of a hemocytometer as adjunct to a subculturing exercise. The
following review information may assist you in proceeding through the exercise.

1. Cell populations are
usually expressed as # cells/ml or # cells/culture.

2. When viewed with a
compound microscope with a 10x ocular and 10x objective (total magnification
100x), one large square of the hemocytometer (1 mm x 1 mm) will fill the field.

3. Each of the four large
corner squares and the large center square of a Neu­bauer type hemocytometer
usually are counted. When the cell count is low (less than 10 cells/square) all
nine squares should be counted.

4. Each large square
measures 1 x 1 mm and is 0.1 mm deep. Hence, each large square has a volume of
0.1 mm3 or 0.001 cm3 or 0.0001 ml (10-4 ml).

5. The final calculation
takes into account:

a. how you wish to express
your count (cells/ml or cells/flask)

b. the dilution (# of mls of
saline or dye or medium into which your cells have been suspended

c. the number of squares
counted

Hence the number of cells/ml of
sample is calculated as follows:

total # cells counted x dilution x 104

number of squares
counted

PROCEDURE:

1. Proceed to make a 1:3
split of a culture following the procedure described above. In order to
determine the number of cells which were harvested from the flask.

2. Aseptically remove 0.5 ml
of cell suspension in trypsin and place in a microfuge tube on ice. Sterility
need not be main­tained.

3. Remove a few drops of the
cell suspension with a Pasteur pipette and load both chambers after putting the
hemocy­tometer coverslip in place.

4. Count the total
number of cells in each of the four corner and central squares.

CALCULATIONS:

1. Count 10 squares and then
multiply that number by 1,000 and that gives us the number of cells per ml from
your solution of cells in trypsin.

N = Sum of 10 Squares X 103

2. This gives you the number
of cells per ml, you should now adjust this for any dilution you are doing. That
is, you have a T-25 flask that you have just added 3.3 ml of trysin to, this is
our total volume of cells and you have calculated the number of cells per ml.
You have removed 0.3 ml (or whatever volume you have used), so you have 3.0 ml
of cells left that you are going to add to a T-25 with 9.0 ml of fresh media.
This gives you a 1:10 dilution of the cells you have just counted.

OK, we have
some data, let's see how good it is. Or more accurately, how precise is our
data. Precise is defined in a statistics as the closeness of repeated
measurements. We will now load the hemocytometer and count 5 squares per side
(total of 10 squares) two times.

This will give
us two groups of 10 readings (each reading represents one square). I want to
know if the means of these two groups of 10 readings are significantly different
at the 0.05 level of significance (alpha is equal to 0.05). Generate a null
hypothesis to test this notion and test for significance.

5. Much of what I have
presented is taken from a course I had in In Vitro Toxicology
at the Center For Advanced Training in Cell and Molecular Biology at Catholic
University, Washington, D.C. 1986.

LAB 6 AND 7

IN VITRO
INVESTIGATIONS OF ZF-4 CELLS

OK, we have
hopefully mastered some skills in cell culture techniques and we have observed
the growth of ZF-4 cells in culture. This weeks lab will be an experiment that
your group has designed to study the effect of exogenous agents on ZF-4 cells.
The students in each group must work together to design and perform your
experiment. Remember, the design of your experiment MUST BE COMPLETED AND
CLEARED WITH ME PRIOR THE SCHEDULED LAB (see syllabus). You will be provided
with the following materials for the laboratory. Other materials not listed may
be made available upon special request.